5887911Southern Research Specialized Biocontainment Screening CenterRSV_TR_52012930A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) (5)Currently, there are no commercially available vaccines to protect humans against Respiratory syncytial virus (RSV). RSV is associated with substantial morbidity and mortality and is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Nevertheless, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. The existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis(R) from MedImmune) that is limited to use in high risk pediatric patients. The economic impact of RSV infections due to hospitalizations and indirect medical costs is greater than $ 650 million annually. The assay provider has developed and validated a plaque assay to confirm antiviral compound effect and determine the potency of lead compounds. We anticipate that the proposed studies utilizing the Molecular Libraries Probes Production Network (MLPCN) HTS resources will generate multiple scaffolds targeting various junctures in the RSV viral lifecycle. These may be furthered developed into probes to construct novel single or combination therapeutics. Cell Culture: HEp-2 cells (ATCC CCL-23, American Tissue Culture Type) were maintained as adherent cell lines in Optimem 1 with 2 mM L-glutamine and 10% fetal bovine serum (FBS) at 37 degrees Celsius in a humidified 5% CO2 atmosphere. Cells were passaged as needed and harvested from flasks using 0.05% trypsin-EDTA. Assay Media - Preparation of Complete DMEM/F12: 50 mL Pen/Strep/Glutamine (Gibco, Cat. No. 10378) was added to four liters of room temperature DMEM/F12 (Sigma, Cat. No. D6434) and the pH adjusted to 7.5 using 1N NaOH. The medium was sterile filtered through a 0.2 um filter and 10 mL of HI-FBS was added per 500 mL of media.Compound Preparation: For plaque assays, compounds or carrier control (DMSO) were diluted in complete DMEM/F12 and 2 mL per well was dispensed to 6-well assay plates (final plate well concentration was 25 micro-molar and final DMSO concentration 0.5%).Avicel Overlay Prep 2X DMEM/F12, 2% Avicel, Pene/Strep, FBS.Control Drug: The positive control drug for this assay, ribavirin [1] (No. 196066, MP Biomedicals, Solon, OH) was solubilized in DMSO. It was diluted and added to the assay plates as described for test compounds. Final concentration for ribavirin was 35 micro-molar. All wells contained 0.5% DMSO.Preparation of HEp-2 cells: Cells were harvested and resuspended to 500,000 cells per mL in For six well plates the cells were suspended in C-Optimem 1 (10% FBS) at 500K/mL and incubated overnight. Assay Set up: HEp-2 cells were seeded in 6 well tissue culture plates at 1,000,000 cells per well in 2 mL Complete Optimem1 and incubated 24 hours at 37 degrees Celsius, 5% CO2. The media was aspirated from the wells, 0.5 mL RSV Long strain (MOI of 0.1) diluted using C-DMEM/F12 was added and the plates incubated at 37 degrees C, 5% CO2, rotating every 20 min. to facilitate infection. After 2 hours, the virus supernatant was aspirated and each well was washed with 3 mL of 1X PBS. Compounds were diluted in Complete DMEM/F12 media to give a final concentration of 25 micro-molar, added to assay plates and incubated at 37 degrees C, 5% CO2 and 90% relative humidity. After 48 hours, the supernatant (RSV/compound/media ; 1.6 mL) was removed, flash frozen on dry ice, and stored at -80 degrees C.HEp-2 cells in Complete Optimem1were seeded in 24 well tissue culture plates at 400,000 cells per well in 0.5 mL and incubated 24 hours at 37 degrees Celsius, 5% CO2. The supernatant (RSV/compound/media) was removed from -80 degrees C and thawed on ice. The supernatants were serially diluted in Complete DMEM/F12 media (1E-00 to 1E-04). The media was aspirated from the 24 well plates, 0.2 mL of each supernatant dilution was added to each well and the plates incubated at 37 degrees C, 5% CO2, rotating every 20 min. to facilitate infection. After 2 hours, 1X PBS was added to each well to wash and aspirated followed by the addition of 0.5 mL 1% Avicel per well. The assay plates were incubated for six days at 37 degrees C, 5% CO2 and 90% relative humidity. Endpoint Read: Following the six day incubation period, the Avicel overlay was aspirated, washed with 0.5mL of PBS, and added 0.5mL of 4% Paraformaldehyde per well. The assay plates were incubated at 4 degrees C for 24 hours. The paraformaldehyde was aspirated, each well washed with 1mL deionized water, and stained with 1mL of 0.05% neutral red with periodic shaking for 10 minutes at room temperature The neutral red was aspirated and the plates were briefly inverted without lids on paper towels for drying.Data Analysis: PFUs were calculated in a manner analogous to the Reed & Muench method of TCID50 determination.Possible artifacts in this assay include, but are not limited to, compounds that precipitate.Scoring: Compounds showing more than 10 fold reduction in the progeny titer (<-1 log virus titer change), and were defined as Active. Scoring for this assay was based on the decreasing in virus titer relative to the untreated, infected control.2391Primary and Confirmatory Screens for inhibitors of Respiratory Syncytial Virus induced cytopathic effect488972Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect492966Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect493016Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect493088Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect504526Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect504655Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect504820Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect504823Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect504827Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect540264Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect540337Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect588485Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect588455Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect2410Counterscreen to identify cytotoxic compounds488976Counterscreen to identify cytotoxic compounds492968Counterscreen to identify cytotoxic compounds493015Counterscreen to identify cytotoxic compounds493090Counterscreen to identify cytotoxic compounds504509Counterscreen to identify cytotoxic compounds504674Counterscreen to identify cytotoxic compounds504818Counterscreen to identify cytotoxic compounds504826Counterscreen to identify cytotoxic compounds504825Counterscreen to identify cytotoxic compounds540266Counterscreen to identify cytotoxic compounds540338Counterscreen to identify cytotoxic compounds588454Counterscreen to identify cytotoxic compounds588483Counterscreen to identify cytotoxic compounds504829Time of Addition449732Plaque Reduction504830Plaque Reduction540320Plaque Reduction588713Plaque Reduction2440Summary AID12814http://www.southernresearch.orgSouthern Research home page1Log virus titer change @ 10 uM12542Log virus titer change @ 5 uM12543Log virus titer change @ 2.5 uM1254101R03 MH084847-01212501142712901-3.3982-1.13130.13820111115125011428121001-5.7542-5.7543-3.0552011111512501143012881-2.7162-0.57230.1552011111512501143111010.14420.26730.2120111115